The present invention relates to aqueous compositions useful for stabilizing polypeptides and antigens. The stabilized polypeptides and antigens are useful in analytic methods such as antigen-specific detection, as well as other pharmaceutical uses where the stabilization of such components in an aqueous solution is desirable.
The following is a discussion of literature potentially relevant to the invention disclosed herein. However, none of the references discussed herein is admitted to be prior art.
Stabilizing polypeptides and antigens in aqueous solutions is often difficult. For example, storage of such solutions at room temperature for prolonged periods results in deterioration of polypeptides or antigens contained in the aqueous solution. In particular, antigens of bacteria, viruses, and other microorganisms have been documented to be unstable when stored in an aqueous medium. One example is enveloped viruses, such as influenza viruses of the Orthomyxovirus family that contain antigens that degrade in solution over time at a broad range of storage temperatures. Those of ordinary skill in the art understand that stabilization in solution of various bacterial antigens, for example, some toxins, is also difficult. To avoid deterioration in an aqueous solution those skilled in the art have used lyophilization and freezing as methods for preserving polypeptides or antigens. Indeed, the widespread use of lyophilization and freezing demonstrates the shortcomings and difficulties of preserving such components in aqueous solutions.
Numerous publications in the art disclose means of increasing the stability of a lyophilized reagent, and the difficulties inherent even within this state of the art technology. For example, U.S. Pat. Nos. 5,955,448, 4,496,537, and PCT filing WO 97/04801 all disclose improvements in lyophilization techniques. Lyophilization of protein containing solutions, however, imposes major costs and inconvenience on the manufacturer and the end user, as well as introducing an increased risk for reconstitution errors and contamination. Additionally, freezing of a solution requires special equipment and ultimately can lead to protein degradation when repeated cycling occurs. For commercial use, deterioration of polypeptides and antigens in aqueous solutions is costly because such solutions require replacement after only a short storage life.
This invention concerns an aqueous stabilizing reagent or diluent that enhances the stability of polypeptides, and other non-proteinaceous compounds in solution. Antigens such as carbohydrates, proteins, lipoproteins, lipopolysaccharides, polysaccharides, nucleic acids, nucleoproteins, and carbohydrates complexed with proteins, lipids, and other compounds are illustrative examples of the types of antigens that may be stabilized using the current invention. Using novel reagent components, the invention improves the stability of polypeptides and antigens in current commercially available diluents or diluents described in the art. The invention is especially useful for stabilizing antigens and polypeptides used as control reagents in diagnostic assays or other uses that require stable aqueous solutions of such components. The stabilizing diluent of the present invention is useful for stabilizing polypeptides and antigens for storage at about 2xc2x0-8xc2x0 C., room temperature and at temperatures of about 45xc2x0 C. for extended periods of time.
PCT filing No. WO99/15901 discloses a diluent for the stabilization of antigens, in particular, Hepatitis C Virus (HCV) antigens. The HCV diluent comprises a reducing agent to keep the HCV antigens in a reduced form. The publication reports that the inclusion of a reducing agent in a diluent maintained the immunoreactivity of an HCV antigen for up to seven days. The reported diluent further comprises sodium phosphate, pH 6.5 (or other buffer), EDTA (or other chelator), DTT (or other reducing agent), gelatin (or other protein blocking source), ammonium thiocyanate (or other chaotrope), sodium azide (or other preservative) and SDS (or other detergent). However, the inclusion of a reducing agent in the diluent may be ineffective in or deleterious to the stability of many antigens from other microorganisms.
U.S. Pat. No. 4,956,274 concerns techniques for stabilizing peptide fragments from xcex2-galactosidase for use in complementation assays. The solution disclosed in U.S. Pat. No. 4,956,274 contained an ionic surfactant or a surfactant derived from a sugar residue to slow degradation of xcex2-galactosidase peptide fragments. However, the surfactants also denatured the enzyme fragments, and thus had to be removed or neutralized to enable the enzymatic fragments to return to their correct conformation and regain enzymatic activity, indicating that the solution did not stabilize the native form of the protein. The surfactants are neutralized just prior to the assay by using cyclodextrin. Alternatively, the action of the surfactants was masked with a high concentration of serum. Additional components of the disclosed reagent included a chelating agent, buffer, bacteriocide, magnesium or other ions, reducing agents, solubilizing agents such as solvents like ethylene glycol, and nonionic detergents. As those in the art will appreciate denaturing and renaturing proteins or enzyme fragments may damage some antigenic epitopes and render them inactive.
U.S. Pat. No. 5,459,033 describes a solution useful for preventing virus aggregation. The solution was reported to contain N-lauryl sarcosine or other anionic surfactants. The solution was said to enhance stability based on the supposition that virion particles, particularly, hepatitis and herpes viruses aggregate due to hydrophobic attractions thereby decreasing sensitivity. This diluent was not reported to improve or preserve the antigen""s catalytic activity or immunogenicity, but to prevent aggregation. Also, before the solution could be used it reportedly had to be incubated for 15 hours to ten days at 2-35xc2x0 C. to insure consistent stability, adding a major limitation to the manufacturability of the final product.
U.S. Pat. No. 5,660,978 discloses a method of stabilizing an antigen, particularly a labile protein antigen, especially an enzyme, by incorporating into a concentrated solution of antigen (such as serum), an antigen-specific antibody or portions thereof (particularly Fab) to prevent proteolysis or oxidation. Introduction of serum or non-specific IgG would not be expected to provide the desired protection or specificity of protection for such a diluent. Moreover, stabilization was completed prior to placing the antigens in a diluent. In contrast, the current invention is stabilized by the diluent itself. Additionally, the current invention stabilizes a relatively dilute solution from the beginning, not in stages as the patented invention suggests. The use of antibodies to structurally stabilize an antigen would be problematic especially if one or more of the antigenic sites are the target of an immunological assay. Additionally, one skilled in the art would have difficulty consistently producing a diluent such as the one disclosed in the ""978 patent, which conformationally preserves the antigen, but does not inhibit specific enzyme activity. The invention is essentially utilizing the antibody portions as a fixative reagent, in place of a chemical fixative and thus, does not truly describe a stabilizing diluent.
Landi, S and Held, H R (Tubercle 59 (1978) 121-133) reported the addition of TWEEN(copyright) 20 (Polyoxyethylene Sorbitan Monooleate), detergent into a diluted solution and suggested that tuberculin PPD stability is was enhanced by this addition due to the detergent""s anti-adsorptive properties. The tuberculin preparation, made by Connaught Laboratories, LTD., contains tuberculin PPD, 0.3% phenol (reported to act as a preservative), and 0.0005% TWEEN(copyright) 80 (Polyoxyethylene Sorbitan Monooleate) in PBS. Phenol is a hazardous material that would be unacceptable in the current invention and is likely to denature some proteins.
Despite advances in diluent formulation and lyophilization storage techniques, the need still remains for a diluent that adequately improves the long-term stability of various polypeptides and antigens, especially antigens from microorganisms, stored in solution.
The present invention features a reagent for stabilizing polypeptides and antigens. The reagent is especially useful for the stabilization of control or reference antigens used in analytical procedures. Antigens such as carbohydrates, proteins, polypeptides, polypeptide fragments, lipoproteins, lipopolysaccharides, polysaccharides, nucleic acids, nucleoproteins, and carbohydrates complexed with polypeptides, lipids, and other compounds are illustrative examples of the types of antigens that may be stabilized using the current invention.
The disclosed reagent surpasses previous formulations for polypeptide and antigen stability at both low and elevated temperatures. Polypeptides and antigens can be stored in the reagent in soluble form for extended periods of time at a variety of temperatures from about 0.5xc2x0 C. to more than about 50xc2x0 C., preferably from about 2-8xc2x0 C., about room temperature (typically from about 23xc2x0 C. to about 28xc2x0 C., with 25xc2x0 C. being particularly preferred) and about 42xc2x0 C. to about 43xc2x0 C., especially about 45xc2x0 C. Stability at 45xc2x0 C. is predictive of the reagent""s ability to provide long-term antigen stability. Additionally, the disclosed reagent has the advantage of being a single aqueous solution for the purpose of stabilizing polypeptides and antigens.
In a first aspect, the invention features an aqueous reagent composition to enhance polypeptide or antigen stability. In certain preferred embodiments, the reagent comprises one or more of the following: buffer(s), blocking agent(s), solvent(s), salt(s), chelator(s), detergent(s), and preservative(s). Preferably, the reagent does not include N-dodecanoyl-N-methylglycine or decanoyl-N-methylgluconamide. The reagent may also comprise components such as tissue culture medium or commercially available diluents.
The term xe2x80x9cbufferxe2x80x9d as used herein refers to compositions well known to the skilled artisan that act to minimize the change in pH of a solution. Preferred buffers have a pKa that provides effective buffering at a pH of between 7 and 9. Preferred buffers are ACES, ADA, BES, bicine, bis-tris, CAPS, CHES, diethylmalonate, glycylglycine, glycinamide HCl, HEPES, HEPPS, imidazole, MES, MOPS, PIPES, POPSO, TAPSO, TES, tricine, tris, bicarbonate, and borate. Particularly preferred are phosphate buffers. Preferred buffering agent concentrations are less than 2 M; most preferred concentrations are 0.2 M, 0.1 M, 0.05 M, 0.05 M, 0.02 M, 0.01 M, 0.005, 0.001, and 0.0001 M, having a pH of 7, 7.25, 7.5, 7.75, 8, 8.25, 8.5, 8.75, and 9.
As used herein, the term xe2x80x9cblocking agentxe2x80x9d refers to a protein rich source containing a mixture of proteins and/or polypeptides, and that may include one or more additional components such as lipids, carbohydrates, salts, and cofactors such as heme. The term xe2x80x9cprotein richxe2x80x9d is defined herein. Such blocking agents can be used to stabilize one or more proteins, polypeptides, and/or antigens in the compositions and methods described herein. Preferred blocking agents have a pH of between about 6.5 and about 8.0, and/or an osmolality of between about 250 and about 350 mOsm/Kg H2O. Particularly preferred blocking agents are sera, such as horse serum, newborn calf serum, calf serum, adult bovine serum, human serum, rabbit serum, sheep serum, etc., and serum replacements known to the skilled artisan. Most preferred as a blocking agent is fetal calf serum.
The term xe2x80x9cprotein richxe2x80x9d as used herein refers to a solution containing a mixture of proteins and/or polypeptides, and having a total protein concentration between about 1 g % and about 50 g %, most preferably between about 3 g % and about 10 g %. For example, fetal calf serum has a total protein content of between about 3 g % and about 4.5 g %, while adult bovine serum has a total protein content of between about 4.5 g % and about 8.5 g %.
The terms xe2x80x9csolventxe2x80x9d and xe2x80x9csolubilizing agentxe2x80x9d as used herein refer to a liquid substance capable of dispersing the other components of the composition. Preferred solvents are water, glycerol, DMSO, alcohols such as ethanol, methanol, etc., acetone, dimethyl sulfoxide, acetonitrile, and dimethyl formamide.
The term xe2x80x9csaltxe2x80x9d as used herein refers to one or more compounds that result from replacement of part or all of the acidic hydrogen of an acid by a metal, or an element acting like a metal. Preferred salts are KCl, NaCl, MgCl2, MgSO4, and CaCl2. Preferred salt concentrations are between 4M and 0.1 mM, most preferably between 2M and 50 mM.
The term xe2x80x9cchelatorxe2x80x9d as used herein refers to a molecule that binds metal ions, usually by binding to two or more complexing groups within the molecule. Chelators are well known in the art, and include certain proteins and polypeptides, as well as small molecules such as ethylenediaminetetraacetic acid (EDTA) and ethylene glycol-bis(xcex2-aminoethyl ether)-N,N,Nxe2x80x2,Nxe2x80x2-tetraacetic acid (EGTA). Preferred chelator concentrations are between 100 mM and 0.01 mM, most preferably between 20 mM and 1 mM.
The term xe2x80x9cdetergentxe2x80x9d as used herein refers to compounds well known in the art that are able to emulsify oils and act as wetting agents. Preferred detergents include CHAPS, cholic acid, deoxycholic acid, digitonin, n-dodecyl-xcex2-D-maltoside, glycodeoxycholic acid, n-lauroylsarcosine, lauryl sulfate, saponin, TWEEN(copyright) 20 (Polyoxyethylene Sorbitan Monolaurate), and TWEEN(copyright) 20 (Polyoxyethylene Sorbitan Monolaurate). Preferred detergent concentrations are between of about 0.001% to about 5%. Most preferably, the composition comprises about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 1.0%, and 2%.
The term xe2x80x9cpreservativexe2x80x9d as used herein refers to compounds well known to the skilled artisan that prevent the growth of microorganisms. Preferred preservatives include thimerosal, sorbic acid, BHA, BHT, MICROCIDE II(copyright) (trimethyltetradecylammonium bromide) and antibiotics such as gentamycin, penicillin, streptomycin, etc. Preferred preservative concentrations are between 10% and 0.001 mM, most preferably between 1% and 0.1%.
As used herein xe2x80x9cpolypeptidexe2x80x9d refers to a polymer of amino acids and does not refer to a specific length of the product, thus, peptides, oligopeptides, proteins, and fragments thereof are included within the definition. The term xe2x80x9cpolypeptidexe2x80x9d does not exclude, post-translational modifications of the polypeptide, for example, glycosylation, acetylation, phosphorylation, and the like.
As used herein xe2x80x9cantigensxe2x80x9d include carbohydrates, proteins, polypeptides, lipoproteins, lipopolysaccharides, polysaccharides, nucleic acids, and carbohydrates complexed with polypeptides, lipids, and other compounds. Antigens may be capable of eliciting an immune response in an animal having a functional immune system. In a preferred embodiment, the antigens stabilized are polypeptide antigens. These polypeptide antigens may be found alone or in association with other molecules. In a particularly preferred embodiment, the polypeptide stabilized are nucleoprotein antigens. Nucleoprotein antigens may be from any number of sources and may be found alone or in association with other molecules.
By xe2x80x9cnucleoprotein antigensxe2x80x9d is meant any polypeptide found associated with or attached to a nuclear complex.
A particularly preferred nucleoprotein is a nucleoprotein from influenza virus, particularly influenza type B.
By xe2x80x9cantigen stabilityxe2x80x9d is meant the ability to maintain a consistent signal generated by one reagent, in an assay or analytical method, especially a diagnostic assay or an immunoassay, after reagent storage for a given period of time at a set temperature. Typical storage temperatures are from about 2xc2x0-30xc2x0 C. Antigen stability can also be assessed by measuring antigen deterioration at elevated temperatures for a shorter period of time. Typical temperatures for accelerated stability tests are from about 30xc2x0 to 60xc2x0 C.
By xe2x80x9cmicroorganismsxe2x80x9d is meant a prokaryote, eukaryote such as yeast, virus, prion or other infectious particle.
By xe2x80x9canalytical methodxe2x80x9d is meant any technique that allows specific detection of one or more antigens. Analytical methods include immunoassays of any detection format and nucleic acid hybridization, numerous examples of which are known in the art. A particularly preferred optical immunoassay method is described in U.S. Pat. Nos. 5,550,063; 5,955,377; and 5,541,057.
In a second aspect, the invention features an aqueous reagent composition to enhance antigen or polypeptide stability wherein the antigen is from a microorganism. In a preferred embodiment, the microorganisms comprise viruses and/or bacteria. The invention is particularly preferred for use with viral analytes. In a most particularly preferred embodiment, the invention features a reagent composition for stabilizing antigens and polypeptides from influenza, especially polypeptides and antigens from influenza B.
In a third aspect, the invention features a reagent composition for stabilizing a nucleoprotein of influenza virus, particularly nucleoprotein from influenza B.
In a fourth aspect, the invention features an aqueous reagent composition for stabilizing an antigen preparation to be used as a control or reference reagent associated with an analytical method.
In a particularly preferred embodiment, the reagent comprises a buffer, a blocking agent, a salt, a chelator, a solubilizing agent, a non-ionic detergent, and a preservative. Most preferably, the reagent does not contain N-dodecanoyl-N-methylglycine or decanoyl N-methylgluconamide.
In further preferred embodiments, the reagent may also contain tissue culture media, STABILCOAT(copyright) buffer (an aqueous solution containing purified bovine protein and other non-toxic chemicals in phosphate buffered saline, pH 7.0-7.4; BSI, Inc.), and formalin-inactivated virus-containing cell culture media.
In yet other preferred embodiments, the reagent comprises sodium phosphate buffer, fetal calf serum, glycerol, sodium chloride, EDTA, TWEEN(copyright) 20 detergent (polyoxyethylene sorbitan monolaurate), MICROCIDE II(copyright) (trimethyltetradecylammonium bromide). Gentamycin Sulfate, and may contain sucrose, tissue culture media and STABILCOAT(copyright) buffer (an aqueous solution containing purified bivine protein and other non-toxic chemicals in phosphate buffered saline, pH 7.0-7.4; BSI, Inc.). The pH of the solution is between about 7 and about 9, most preferably between 7.5 and 8.5. One skilled in the art understands that similar reagents can substitute for those listed above. For example, EDTA can be replaced with EGTA, and MICROCIDE II(copyright) (trimethyltetradecylammonium bromide), or gentamycin can be replaced by other anti-bacterial agents. Preferred concentrations of the above listed reagents are as follows: for sodium phosphate, 0.1 mM to 1000 mM, more preferably 1 mM to 200 mM, most preferably 50 mM to 100 mM; for fetal calf serum, 0.1% to 40% v/v, most preferably 2% to 20% v/v; for glycerol, 0.1% to 30% v/v, most preferably 2.5% to 10% v/v; for sodium chloride, 0.1 mM to 4 M, most preferably 50 mM to 2 M; for EDTA, 0.01 mM to 100 mM, more preferably 1 mM to 20 mM, most preferably 10 mM to 15 mM; for TWEEN(copyright) 20 (Polyoxyethylene Sorbitan Monolaurate), 0.001% to 1%, more preferably 0.01% to 0.1%, most preferably 0.5%; for MICROCIDE II(copyright) (trimethyltetradecylammonium bromide), 0.001% to 1% w/v, most preferably 0.002% to 0.1% w/v; for gentamycin sulfate, 0.01% to 10% w/v; more preferably 0.1% to 2.5%, most preferably 0.25% to 1%; and for sucrose, 0.01% to 5% w/v, most preferably 0.1% to 0.5%.
In especially preferred embodiments, the diluent comprises an amount greater than or equal to about: 50 mM sodium phosphate; 2% v/v fetal calf serum; 10% v/v glycerol; 50 mM sodium chloride; 10 mM EDTA; 0.05% v/v TWEEN(copyright) 20 (Polyoxyethylene Sorbitan Monolaurate) detergent; 0.01% w/v MICROCIDE II(copyright) (trimethyltetradecylammonium bromide) preservative; and 0.5% w/v gentamycin sulfate. The reagent may also comprise up to 0.5% sucrose, 15% STABILCOAT(copyright) buffer (an aqueous solution containing purified bovine protein and other non-toxic chemicals in phosphate buffered saline, pH 7.0-7.4; BSI, Inc.) and 20% tissue culture medium from the antigen preparation or by separate addition. The preferred pH of the solution is between about 7.5 to about 8.5. It may also be possible to replace the buffer and/or salt with a concentration of pre-formulated tissue culture medium (for example, Eagle""s Minimum Essential Media or Dulbecco""s Modified Eagle Media from BioWhittaker).
In a fifth aspect, the invention features methods for stabilizing polypeptides and antigens derived from microorganisms. In a preferred embodiment said method is used for stabilizing an antigen preparation from a microorganism for use as a control or reference reagent associated with an analytical method or for use in a pharmaceutical preparation.